N. G. Kolosova

An attempt to prevent senescence: A mitochondrial approach

Antioxidants specifically addressed to mitochondria have been studied to determine if they can decelerate senescence of organisms. For this purpose, a project has been established with participation of several research groups from Russia and some other countries. This paper summarizes the first results of the project. A new type of compounds (SkQs) comprising plastoquinone (an antioxidant moiety), a penetrating cation, and a decane or pentane linker has been synthesized. Using planar bilayer phospholipid membrane (BLM), we selected SkQ derivatives with the highest permeability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decyl-rhodamine 19 (SkQR1), and methylplastoquinonyldecyltriphenylphosphonium (SkQ3). Anti- and prooxidant properties of these substances and also of ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested in aqueous solution, detergent micelles, liposomes, BLM, isolated mitochondria, and cell cultures. In mitochondria, micromolar cationic quinone derivatives were found to be prooxidants, but at lower (sub-micromolar) concentrations they displayed antioxidant activity that decreases in the series SkQ1=SkQR1>SkQ3>MitoQ. SkQ1 was reduced by mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Nanomolar SkQ1 specifically prevented oxidation of mitochondrial cardiolipin. In cell cultures, SkQR1, a fluorescent SkQ derivative, stained only one type of organelles, namely mitochondria. Extremely low concentrations of SkQ1 or SkQR1 arrested H(2)O(2)-induced apoptosis in human fibroblasts and HeLa cells. Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In the fungus Podospora anserina, the crustacean Ceriodaphnia affinis, Drosophila, and mice, SkQ1 prolonged lifespan, being especially effective at early and middle stages of aging. In mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, canities, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc. SkQ1 manifested a strong therapeutic action on some already pronounced retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM SkQ1, vision was restored to 67 of 89 animals (dogs, cats, and horses) that became blind because of a retinopathy. Instillation of SkQ1-containing drops prevented the loss of sight in rabbits with experimental uveitis and restored vision to animals that had already become blind. A favorable effect of the same drops was also achieved in experimental glaucoma in rabbits. Moreover, the SkQ1 pretreatment of rats significantly decreased the H(2)O(2) or ischemia-induced arrhythmia of the isolated heart. SkQs strongly reduced the damaged area in myocardial infarction or stroke and prevented the death of animals from kidney ischemia. In p53(-/-) mice, 5 nmol/kgxday SkQ1 decreased the ROS level in the spleen and inhibited appearance of lymphomas to the same degree as million-fold higher concentration of conventional antioxidant NAC. Thus, SkQs look promising as potential tools for treatment of senescence and age-related diseases.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 4. Age-related eye disease. SkQ1 returns vision to blind animals

Mitochondria-targeted cationic plastoquinone derivative SkQ1 (10-(6′-plastoquinonyl) decyltriphenylphosphonium) has been investigated as a potential tool for treating a number of ROS-related ocular diseases. In OXYS rats suffering from a ROS-induced progeria, very small amounts of SkQ1 (50 nmol/kg per day) added to food were found to prevent development of age_induced cataract and retinopathies of the eye, lipid peroxidation and protein carbonylation in skeletal muscles, as well as a decrease in bone mineralization. Instillation of drops of 250 nM SkQ1 reversed cataract and retinopathies in 3-12-month-old (but not in 24-month-old) OXYS rats. In rabbits, experimental uveitis and glaucoma were induced by immunization with arrestin and injections of hydroxypropyl methyl cellulose to the eye anterior sector, respectively. Uveitis was found to be prevented or reversed by instillation of 250 nM SkQ1 drops (four drops per day). Development of glaucoma was retarded by drops of 5 μM SkQ1 (one drop daily). SkQ1 was tested in veterinarian practice. A totally of 271 animals (dogs, cats, and horses) suffering from retinopathies, uveitis, conjunctivitis, and cornea diseases were treated with drops of 250 nM SkQ1. In 242 cases, positive therapeutic effect was obvious. Among animals suffering from retinopathies, 89 were blind. In 67 cases, vision returned after SkQ1 treatment. In ex vivo studies of cultivated posterior retina sector, it was found that 20 nM SkQ1 strongly decreased macrophagal transformation of the retinal pigmented epithelial cells, an effect which might explain some of the above SkQ1 activities. It is concluded that low concentrations of SkQ1 are promising in treating retinopathies, cataract, uveitis, glaucoma, and some other ocular diseases.

Long-term antioxidant supplementation attenuates oxidative stress markers and cognitive deficits in senescent-accelerated OXYS rats

Oxidative damage of biomolecules increases with age and is postulated to be a major causal factor of various neurodegenerative disorders. Consequently, the concept of neuroprotection by antioxidants has been developed. Recently we have shown that the behaviour of young senescent-accelerated OXYS rats is similar to the behaviour of old Wistar animals. To determine the role of oxidative stress in this phenomenon we investigated age-related changes in protein carbonyls (PrC), lipid peroxides (LP), reduced glutathione (GSH), alpha-tocopherol (TP) and SOD activity in the brain of OXYS and Wistar rats. We also studied the effect of long-term supplementation with bilberry extract (2g/kg of diet) and Vitamin E (140 mg/kg of diet) on oxidative stress markers and on learning in passive avoidance test. In both rat strains LP, PrC and TP increased with age and at 24 months PrC was significantly higher (p<0.0001) in OXYS rats. At 3 months GSH was higher and SOD activity was lower in OXYS rats than in Wistar rats. SOD activity decreased with age in OXYS whereas increased in Wistar rats. Cognitive impairments in OXYS rats were manifested earlier than significant differences in the level of brain oxidative stress markers between two strains. By contrast, differences in antioxidant systems of Wistar and OXYS rats were registered at 3 months. Antioxidants attenuated cognitive deficits in OXYS rats, providing evidence for therapeutic role of antioxidants. Nevertheless, the exact mechanisms of neuroprotective effects of antioxidants in vivo and the real impact of oxidative stress on the development of cognitive impairments in OXYS rats still needs to be further investigated.

Clinical and Morphological Characteristics of Chorioretinal Degeneration in Early Aging OXYS Rats

OXYS rats are characterized by early development of cataract and chorioretinal degeneration with clinical manifestations similar to those observed in senile cataract and ageassociated macular degeneration in humans. According to fundoscopy findings, the incidence of chorioretinal degeneration sharply increases in OXYS rats by the age of 4.5 months, when all animals develop signs of fundus oculi pathology. Morphological analysis of semithin sections of the posterior wall of the eye in OXYS rats aged 5 months showed that choroid vessels, pigmented epithelium, and radial glia were most vulnerable to injury. Retinal hypoxia and destruction of the pigmented epithelium associated with circulatory disorders in the choroid vessels presumably lead to injuries of the neurosensory cells (mainly the external segments) and a 3.5-fold increase in the percent of photoreceptors with nuclear pyknosis in comparison with the control. These results indicate that OXYS rats represent an adequate model of age-associated macular degeneration and can be used for studies of the pathogenesis of this condition and development of methods for its treatment and prevention.

Changes in physicochemical parameters and α-crystallin expression in the lens during cataract development in OXYS rats

The pathogenesis of cataract is associated with oxidative stress and with altered crystallin expression but it is still understood incompletely. In this study, the senescence-accelerated OXYS rats were used as a model. The first biomicro-scopic signs of cataract in OXYS rats were registered at the age of 1.5 months; at 3 months morbidity reached 90%, and at 6 months it reached 100%. Cataract manifestation progresses: at 24 months mature cataract was detected in 90% of eyes of OXYS rats, whereas in 80% of Wistar rat eyes only initial signs of this disease were detected. Analysis of lens redox-parameters has shown that in OXYS rats the intensity of tryptophan fluorescence is higher, the GSH content being higher at 2 months but during formation of mature cataract at 13, 18, and 24 months being lower than in Wistar rats. Decrease in solubility of OXYS rat lens proteins was observed at the age of 13 months. At the age of 3 months gene expression of αA-crystallin and αB-crystallin was 3-fold and 25% lower, respectively, than in Wistar rats. At the age of 14 months there was a 27-fold decrease in expression of αB-crystallin in OXYS rats and it became 21-fold lower than in control. Proteins are synthesized in lens epithelial cells and dystrophic changes in senile cataract result in decrease in structural protein expression. The changes observed in OXYS rats are evidently associated with the dystrophic changes in lens epithelium, which we have described earlier, and are consistent with the model of senile cataract.

Effect of Histochrome on Brain Vessels and Research and Exploratory Activity of Senescence-Accelerated OXYS Rats

Changes in the diameter of brain vessels and intensity of collateral blood flow typical of chronic ischemia were detected by magnetic resonance imaging in senescence-accelerated OXYS rats demonstrating reduced (compared to Wistar rats) research and exploratory activity. Histochrome (antioxidant drug) produced positive effects on cerebral vessels in OXYS rats by stimulating collateral blood flow and acting as a vasodilator agent. Analysis of correlations showed that these effects of histochrome were closely related to its capacity to activate research and exploratory activity and reduce anxiety of OXYS rats in the open field test.

Lipofuscin granule dynamics during development of age-related macular degeneration.

The pigment epithelium cell structure and therapeutic effect of antioxidant SkQ1, selectively penetrating into mitochondria from eye drops, were studied upon development in OXYS rats of age-related retinopathy as a model of macular degeneration. The characteristic dynamics and ultrastructural peculiarities of the layer of electron-dense cytoplasmic structures of the pigment epithelium apex part and incorporated lipofuscin granules were revealed. The therapy of OXYS animals for 68 days using 250 nM SkQ1 drops decreased the extent of development of age-related macular degeneration. Electron-microscopic investigation showed that SkQ1 prevented development of ultrastructural changes in the pigment epithelium characteristic of macular degeneration, the condition of which after therapy with SkQ1 drops corresponded to ultrastructure of pigment epithelium in Wistar rats of the same age having no symptoms of retinal damage. It is supposed that ultrastructural changes in the electron-dense layer upon development of age-related macular degeneration are indicative of disturbances in the optical cycle functioning, especially of disturbances in functioning of photoreceptor membranes.

Effects of the mitochondria-targeted antioxidant SkQ1 on sexually motivated behavior in male rats

Ample research indicates that age-related neuronal-behavioral decrements are the result of oxidative stress and may be ameliorated by antioxidants. Here we examined effects of mitochondria-targeted antioxidant, SkQ1, on sexual motivation in 12-month-old Wistar and accelerated-senescent OXYS male rats. A change in behavioral activity of a male at a holed transparent partition with a receptive female on the other side was taken as an index of sexual motivation. The social behavior of male in same conditions with ovariectomised (OVXed) female and castrated male was investigated to differentiate sexually and socially motivated behavior. Behavioral response to social stimulus did not depend on age and genotype. No differences were found between 4- and 12-month-old Wistar males when sexual stimulus was presented; however, 12-month-old OXYS males demonstrated a lower propensity for sexual motivation as compared to 4-month-old OXYS rats and 12-month-old Wistar rats. We examined effects of SkQ1 on sexual motivation in 12-month-old male rats following prolonged supplementation begun at 1.5months of age (10, 50 or 250nmol/kg daily), a 45-day supplementation begun at 10.5months of age (50nmol/kg) and a 3-month supplementation begun at 9months of age (250nmol/kg). SkQ1 did not affect locomotor activity; however, it increased the time spent at the partition. A significantly higher measure of the motivational stage of sexual behavior was displayed following chronic preventive treatment at a dose of 50 and 250nmol/kg by OXYS rats. Chronic therapeutic treatment during 3months at a dose of 250nmol/kg was effective in age-accelerated OXYS rats too. These findings suggest an essential role for oxidative stress associated with mitochondrial dysfunction in the decline of sexually motivated behavior of male rats. Recovery from these impairments and/or their prevention enables a fully successful performance of the initial stage of male sexual behavior.

Opposite effects of antioxidants on anxiety in Wistar and OXYS rats

Passive behavior in the open field test and high anxiety in an elevated plus-maze develop in OXYS rats by the age of 3 months and are regarded as manifestations of early aging. We studied the possibility of preventing these disturbances with vitamin E and whortleberry extract (20 mg/kg for 45 days from the age of 1.5 months). Whortleberry extract alone increased horizontal activity and reduced anxiety of OXYS rats, while anxiety in Wistar rats increased significantly after treatment with both preparations, and especially with vitamin E: the number of entries into open arms of the elevated plus-maze and the time spent there decreased. The results necessitate comprehensive evaluation of the aftereffects of long-term use of antioxidants, acknowledged geroprotectors intended for preventive use.

Evaluation of Effects of Histochrome and Mexidol on Structural and Functional Characteristics of the Brain in Senescence-Accelerated OXYS Rats by Magnetic Resonance Imaging

The effects of histochrome and mexidol on the morphology and function of the brain and behavior were studied in senescence-accelerated OXYS and Wistar rats. MRI showed that signs of neurodegenerative changes were present in OXYS rats at the age of 3 months and were pronounced at the age of 12 months. Histochrome (1 mg/kg, 5 days) more effectively than mexidol (4 mg/kg, 7 days) reduced anxiety and increased exploratory activity of 1-yearold OXYS rats. Both drugs improved the morphology and function of the brain. Their effects consisting in correction of diffuse changes in the white matter and reduction of edema were comparable; in addition, histochrome reduced the intensity of demyelinization processes.